This project studies peripheral blood hematopoietic progenitors (PBHP) as a target for gene therapy of inherited diseases affecting the function of human phagocytic cells, including neutrophils,eosinophils, and monocytes. A related goal of this project is development of novel cellular therapies based on genetically engineering human PBHP and their phagocytic cell progeny to endow them with new properties to augment host defense against chronic infections including tuberculosis and other mycobacteria infections. To achieve these goals it is necessary to define conditions for optimal harvest, purification and culture of the primitive human hematopoietic cells with a CD34 surface antigen phenotype. In a clinical study we determined that 5 ug/kg daily subcutaneous granulocyte colony stimulating factor administered for 5 or 6 daily doses is optimal for recruiting a 20 to 100 fold increase in circulating primitive CD34 progenitor cells. Using apheresis and magnetic bead purification technology over 100 million purified CD34 cells can be harvested from G-CSF recruited volunteers. These cells can be used as a target for gene transfer studies. The recruited cells are more receptive to retrovirus mediated gene transfer than similar CD34 cells from volunteers not treated with G-CSF. In related studies we find that the combination of the growth factors SCF, IL3 and IL6 used to culture these cells preserves the CD34+ primitive phenotype longest in culture. We developed specific culture conditions leading to predominantly neutrophils, eosinophils or monocyte/macrophages. This will be important for future studies aimed at engineering new characteristics into specific end stage phagocytes by gene transfer into progenitors. As part of our studies to achieve gene therapy for the inherited immunodeficiency, chronic granulomatous disease (CGD), a sensitive FACS assay of superoxide/hydrogen peroxide production by mature neutrophils was developed based on the oxidation induced increase in fluorescence of rhodamine-123. Using this assay it was possible to follow the maturation of functional oxidase as a marker of the appearance and number of maturing neutrophils over time in cultures of PBHP. This information is essential for planning and evaluating studies in which genes are transferred into CGD progenitors to correct NADPH oxidase activity as gene therapy for CGD.